Abstract Evolutionary history and taxonomic position for cryptic species may be clarified by using molecular and cytogenetic methods. The subterranean rodent, the Alay mole vole Ellobius alaicus Vorontsov et al., 1969 is one of three sibling species constituting the subgenus Ellobius Fischer, 1814, all of which lost the Y chromosome and obtained isomorphic XX sex chromosomes in both males and females. E. alaicus is evaluated by IUCN as a data deficient species because their distribution, biology, and genetics are almost unknown. We revealed specific karyotypic variability (2n = 52–48) in E. alaicus due to different Robertsonian translocations (Rbs). Two variants of hybrids (2n = 53, different Rbs) with E. tancrei Blasius, 1884 were found at the Northern slopes of the Alay Ridge and in the Naryn district, Kyrgyzstan. We described the sudden change in chromosome numbers from 2n = 50 to 48 and specific karyotype structure for mole voles, which inhabit the entrance to the Alay Valley (Tajikistan), and revealed their affiliation as E. alaicus by cytochrome b and fragments of nuclear XIST and Rspo1 genes sequencing. To date, it is possible to expand the range of E. alaicus from the Alay Valley (South Kyrgyzstan) up to the Ferghana Ridge and the Naryn Basin, Tien Shan at the north-east and to the Pamir-Alay Mountains (Tajikistan) at the west. The closeness of E. tancrei and E. alaicus is supported, whereas specific chromosome and molecular changes, as well as geographic distribution, verified the species status for E. alaicus . The case of Ellobius species accented an unevenness in rates of chromosome and nucleotide changes along with morphological similarity, which is emblematic for cryptic species.